The long term goal of the proposed research is to understand the mechanisms of protein-protein interactions in signal transduction initiated by cell-cell and cell-matrix interactions. We focus on identification and characterization of cellular proteins that interact with a cytoplasmic protein tyrosine kinase FAK which has been implicated as a central player in intracellular signal transduction by ECM and perhaps other stimuli. Consistent with this, we have recently found that FAK can associate with another intracellular signaling molecule, phosphatidylinositol 3-kinase (PI3K). This interaction is regulated by both cell adhesion and platelet-derived growth factor (PDGF). In addition, using the yeast two-hybrid system we have identified a novel cellular protein, Fip1, that also interacts with FAK. In this proposal, we plan to dissect molecular interactions of FAK with PI3K and Fip1 and investigate biological functions of these interactions in signal transduction by using a combination of biochemical, molecular and cell biological approaches. We will identify the sequences on FAK for binding PI3K by using site-directed mutagenesis and in vitro binding assays, examine the role of the binding sites in mediating regulation of association by cell adhesion in vivo, and investigate the potential functional significance of this interaction in regulating PI3K activity using both in vivo and in vitro assays. Next, we will identify additional sequences on FAK (if any besides the primary binding site identified above) involved in increased FAK:PI3K association in response to PDGF stimulation using transfection assays, explore the potential functions of this interaction in mediating cytoskeletal reorganization elicited by PDGF, and finally investigate the possibility that the Rho subfamily of G proteins (RhoA, Rac1 and CDC42Hs) are involved in the interactions. In parallel, we will isolate the full length cDNA encoding Fip1 using the available partial clone to screen human brain cDNA libraries, sequence the entire clone to deduce amino acid sequences for analysis of potential functional domains, and generate polyclonal antisera against Fip1 using fusion proteins produced in bacteria and synthetic peptides. We will then confirm in vivo interaction of the endogenous Fip1 with FAK, examine its regulation by cell adhesion and growth factors, and determine the FAK-binding domain on Fip1. Finally, we plan to identify additional FAK-interacting proteins by the yeast two-hybrid system. Together these studies will enhance our understanding of the molecular components and mechanisms of signal transduction by ECM which are critical factors both in normal development and in various diseases including cancer. Knowledge gained from these studies may provide novel targets and/or strategies for rational drug design based on the structures and actions of the signaling molecules.